It has long been pointed out that asparagine is essential (an essential amino acid) for the growth of leukemic cells. Extensive studies have been on the therapy of leukemia by eliminating asparagine which is not necessarily essential (non-essential) for normal cells from blood, which therapy is expected to cause no damage to normal cells (L. T. Mashburn et al., Biochem. Biophys. Res. Commun., 12, 50 (1963)). Whereas normal cells in which asparagine is formed from aspartic acid or aspartates with asparagine synthetase, an asparagine-synthesizing enzyme, leukemic cells which is deficient in the asparagine-synthesizing activity due to tumorigenesis and in which no asparagine is formed make use of the asparagine existing in plasma in a very small amount for the protein synthesis. Therefore, the therapy is based upon the idea that the protein synthesis (growth) of leukemic cells will be inhibited by introducing asparaginase into blood of the patient in order to decompose the asparagine in blood (achieve and maintain asparagine deficiency).
It is demonstrated that a variety of asparaginases are useful for the therapy of certain leukemias and solid tumors including acute lymphatic leukemia. This therapy was called attention as a specific and favorable therapeutic idea which represents inhibition (prevention) of the growth of leukemic cells without damaging normal cells (H. Marquardt, Arzneimittel-Forsch., 18, 1380 (1968)). Clinical trials were extensively carried out using asparaginase from Escherichia coli B (R. H. Adamson et al., Cancer Chemother. Rep., (1) 52, 617 (1968)). It was pointed out as a result of the trials that antigen-antibody reaction (immunoreaction) due to administration of a foreign protein in the human body was a problem; side effects such as vomiting, nausea, anorexia, pyrexia, bodyweight decrease, hypohepatia, pancreatitis, oligochromemia, uremia, fibrinogenopenia, hyponoia, skin rash, diarrhea, pararitium, anemia, leukopenia, thrombocytopenia, anaphylaxic shock, cephalalgia, angiodynia, irritation and cramp were observed (P. Laboureur, Pathol. Biol. (Paris), 17, 885 (1969)). Accordingly, the therapeutic method has been considered to be of little practical usefulness despite its causing little damage to normal cells, and, contrary to earlier expectation, there has been applied to some extent combination therapy with chemotherapeutic agents such as prednisone, vincristine, methotrexate, 6-mercaptopurine, cytarabine and cyclophosphamide, and radiotherapy. If there were provided means for avoiding the immunoreaction caused by the foreign protein, it is expected that the therapy will recover great hope. As a means of the solution there has been proposed a scheme in which blood is temporarily drawn out of the body, contacted with immobilized asparaginase (macromolecular material with the enzyme embedded or bound) to decompose the asparagine dissolved in the blood and then returned to the body (extracorporeal circulation) (D. Sampson et al., Trans. Am. Soc. Artif. Intern. Organs, 18, 54 (1972)). In this scheme, however, coagulation of the circulated blood by contact with the enzyme-immobilizing material (macromolecular material) is a newly caused problem, although the immunoreaction with the foreign protein (enzyme) is greatly weakened (or abolished) by employing an adequate enzyme-immobilization technique.
An expedient may be adopted to add an anti-coagulant such as heparin to the blood stream in consideration of poor antithrombogenicity in any of the known enzyme-immobilizing macromolecular materials. Combined use of drugs including heparin in a large amount for a long period of time itself is physiologically undesirable. Therefore, it has been desired to develop macromolecular materials which are capable of not only firmly immobilizing and maintaining asparaginase but also being free of immunoreaction with the enzyme as well as producing no thrombosis by contact with blood stream.
The present invention provides novel antithrombogenic macromolecular materials for immobilizing the enzyme which is suitable for the aforementioned needs.
Thrombosis or deposition of blood components upon the contact surface of a synthetic or natural macromolecular material with blood in medical use has long been recognized as an important problem to be overcome in developing artificial valve, blood vessel, kidney, catheter and the like. Efforts has been continued to find materials which hardly behave as foreign matter to blood, that is, materials which hardly cause thrombus due to destruction of the blood.
A number of attempts have been made to incorporate a minimum amount of an anticoagulant in the surface of a prosthesis based upon an idea that an anticoagulant may not be present throughout the body (circulating blood) in order to prevent coagulation on the contact surface of a prosthesis with blood. For example, application of an anticoagulant such as heparin, hirudin or antithrombin, a platelet-agglutination inhibitor such as adenylcyclase, prostaglandin E or methylxanthine, or a fibrinolysis activator such as urokinase or streptokinase on the surface of a prosthesis, the adsorption via ion-binding functional group, and the fixing on the surface of a prosthesis by means of covalent bond are known. However, the application or adsorption method is disadvantageous in that the anticoagulant and other agents are readily eliminated to have a short effective period of time. The covalent bond method also is not considered as useful, because there is often associated destruction of the anticoagulant by application of the chemical reaction and is possibility of producing adverse reactions to the body by the introduction of covalent functional group; it is also expected that effect of the anticoagulant fixed by means of a covalent bond is not sufficiently high (H. Tanzawa et al., Trans. Am. Soc. Artif. Intern. Organs, 19, 188 (1973)).
In order to avoid such difficulties blending and embedding of an anticoagulant in a prosthesis have been attempted (H. Tanzawa et al., Trans. Am. Soc. Artif. Intern. Organs, 19, 188 (1973)). It is however pointed out that the anticoagulant without fixing (entrapping) treatment by means of a chemical bond is apt to be eluted (released) entirely in a short period of time. The effective period of time is recognized to be 5-8 hours and at longest about 5 days. Therefore, needs for new superior antithrombogenic materials are high.
The present invention provides novel highly antithrombogenic medical materials comprising hydrogels with a high mechanical strength.
The invention provides a process for producing immobilized enzyme preparations by embedding asparaginase in a specific anticoagulant hydrogel.
The invention provides immobilized enzyme preparations in which the subject L-asparaginase is firmly embedded in a medical hydrogel without any damage so as to be sufficiently active because of absence of a conventional chemical bonding process using a chemical reagent or radiation.
In the present invention, polyvinyl alcohol is employed as the starting material for the preparation of an antithrombogenic hydrogel. There have been proposed many methods of the gel formation (preparation of hydrogels) of polyvinyl alcohol. However, as summarized below, all of the methods involve problems in operation or in properties of the product.
(1) By air-drying an aqueous polyvinyl alcohol solution there is obtained a wet or dry film, which, however, is merely a weak film being inferior in resistance to water and having no rigidity in water and is useful in limited applications only (Japanese Patent Publication No. 9523/1965).
(2) Also by adding an acid to an aqueous suspension containing polyvinyl alcohol and tetraethyl silicate, there is obtained a film similar to the one under (1) above only. In this method, addition of an acid to an aqueous solution followed lyophilization is also proposed. The resulting film, however, is of a lower strength and is scarcely moldable (Japanese Patent Publications Nos. 30358/1980 and 11311/1980).
(3) A gelation method involving irradiation of an aqueous polyvinyl alcohol solution with cobalt 60 (.gamma.-ray) is well known. The method, however, not only requires special equipment (facilities for the irradiation) and is high in irradiation cost, but also produces only weak gels which often require an additional hardening process (secondary hardening). Therefore, the gel obtained by this method is of little use except for special applications such as for an artificial vitreous body (intra-eyeball filling liquid) for which a highly viscous liquid (or a soft gel) is desired (J. Material Sci., 1974, 1815 and Japanese Patent Laid Open No. 55647/1975).
(4) Also, it has long been known that an aqueous polyvinyl alcohol solution is gelled promptly upon mixing with boric acid (or an aqueous borax solution) (Note: borax=sodium tetraborate decahydrate). However, the resultant gel is weak and fluid; besides, it is torn immediately when picked up with finger tips so that it is difficult to retain its shape when molded (J. Am. Chem. Soc., 60, 1045 (1938) and French Patent No. 743942 (1933)).
Moreover, although the borax gel is existing in an alkaline condition, it is collapsed at a pH not higher than 8. Therefore, it is hardly usable and of little value for the medical application.
(5) There have also been proposed various gelation methods of polyvinyl alcohol by the use of phenols such as phenol, naphthol or Congo Red, an amino compound or a metallic compound such as a titanium, chromium or zirconium compound. In all of these methods, however, the same drawbacks as in the foregoing (4) are encountered (Japanese Patent Publications Nos. 9523/1965 and 23204/1965).
(6) It is also well known to gel a polyvinyl alcohol using a cross-linking agent or a copolymerization component such as an aldehyde, a dialdehyde, an unsaturated nitrile, a diisocyanate, trimethylolmelamine, epichlorohydrin, bis(.beta.-hydroxyethyl)sulfone, polyacrylic acid, dimethylolurea or maleic anhydride. This method, however, not only requires a process with a chemical reagent but also hardly produces a strong highly hydrous gel (Textile Res. J., (3) 189 (1962) and British Patent No. 742,900 (1958)).
(7) Also, it has long been known to gel an aqueous polyvinyl alcohol solution by allowing it to stand at a low temperature not higher than 40.degree. C., particularly from 5.degree. to 18.degree. C. or lower.
However, the gels formed at room temperature are fragile like agar or carrageenan. Besides, they are dissolved merely by vigorously stirring, stirring after addition of water or slightly warming.
It is also known that it is preferable to employ a low temperature in order to prepare the cooled gel of an aqueous polyvinyl alcohol solution. For example, it is known to carry out the preparation at 18.degree. C., at a low temperature not higher than 0.degree. C. or at a lower temperature (Polymer J., 6, 103 (1974)).
In any case, however, the gel thus obtained is a weak gel (or viscous liquid) like agar, carrageenan or jelly. It is not only very sticky but also is poor in resistance to water so that it is swollen in water to a remarkable extent and further softened; it is partly dissolved out in water and becomes paste-like for the remainder. In water or in warm water at 40.degree.-50.degree. C., the gel is more rapidly deformed and dispersed or dissolved in water. These drawbacks make it hardly valuable for medical use.
(8) Sponge-like product obtained by formalization of polyvinyl alcohol also has long been known. As it is unstable in tissue, in which as it is decomposed or degenerated it produces adverse reactions in the circumstances, the use has recently been very restricted (J. R. Lewis, Plastic & Reconstructive Surgery, 35, 51 (1965)).
(9) It is also known to add a small amount of polyvinyl alcohol to an aqueous solution of a water-soluble macromolecular substance capable of being gelled such as agarose, agar, albumin, alginate, curdlan, carrageenan, casein, CMC, furcellaran, gelatin, methylcellulose, pectin, starch, tamarind gum, xanthan gum, tragacanth gum or guar gum and cool the mixture, immerse it in a gelling agent-containing bath (coagulation bath) or freeze-dry it (Japanese Patent Publications Nos. 25210/1981 and 25211/1981). Also by such method there is obtained only a weak viscous liquid poor in resistance to water, a non-fluid gel or a water-soluble dry powder only.
As a result of studies with an object of developing a process for producing water-insoluble antithrombogenic highly hydrous immobilized enzyme preparations with excellent mechanical properties by the use of polyvinyl alcohol, it was previously found by us that highly hydrous gels with enzyme embedded which had a water content of 45-92% by weight were produced by subjecting an aqueous solution containing polyvinyl alcohol and enzyme to cooling, solidifying and molding at a temperature lower than -15.degree. C. under specified conditions and subsequently, without thawing, to partial dehydration under vacuum (U.S. patent application No. 344,006). The present invention represents further development of the above-mentioned finding. The hydrogels obtained according to the invention does not produce any damage in L-asparaginase, because, in the gelation process and its pretreatment, there is need of none of the acid, alkali, radical source, radiation, organic solvent, reagent and inorganic solvent other than water which are conventionally used in the prior-art gel formation or enzyme immobilization. Moreover, the gels obtained according to the invention are high in water content and are of both rubber-like elasticity and high mechanical strength. The gels of the invention are also insoluble in cold or warm water and non-sticky thereby being entirely differentiated from the aforementioned gels produced by cooling an aqueous solution of polyvinyl alcohol. Thus, the present invention provide a process for preparing L-asparaginase-immobilizing agents by the use of a novel antithrombogenic gel which is entirely different from the prior finding on the gel formation of aqueous polyvinyl alcohol solution by cooling or chemically treating the solution.
This invention is based upon the findings that highly hydrous gels with excellent antithrombogenicity are produced by subjecting a cooled and solidified product obtained from an aqueous solution containing polyvinyl alcohol and asparaginase under specified conditions, without being molten, to a partial dehydration treatment, and the asparaginase embedded (entrapped) in the highly hydrous gel is capable of decomposing and eliminating the asparagine in blood flow without the formation of thrombi on the surface of the gel through which blood is passed.